PROJECT SUMMARY Vector-borne obligate intracellular bacteria are an important cause of morbidity and mortality worldwide. One such pathogen, Orientia tsutsugamushi, causes the neglected tropical disease scrub typhus, which is estimated to cause infection in at least 1 million people each year, and is associated with high mortality if not treated promptly and with the appropriate antibiotics. The species O. tsutsugamushi is characterised by high strain diversity, where different strains exhibit different levels of virulence in patients and animal models. A major factor in the pathogenicity induced by O. tsutsugamushi is an over-activation of the inflammatory immune response. However, the detailed mechanisms describing how O. tsutsugamushi induces this response remain unknown. O. tsutsugamushi is genetically intractable and therefore functional genomics based approaches to identify virulence factors are not possible. Here we propose to take advantage of strain-specific natural variation to uncover key pathways important for virulence. In our preliminary work we have quantified the virulence of two clinical isolate strains of O. tsutsugamushi, Karp and UT176, and shown that they have significantly different outcomes in a mouse model. We have also used dual RNAseq to identify both bacterial and host genes that are differentially expressed between these two strains when grown in human cultured cells (HUVEC), and identified bacterial (surface proteins TSA22, TSA47, TSA56 and Ankyrins Ank6, Ank16, Ank17, Ank20) and host (SOCS2, IL33, other chemokines and cytokines) factors that strongly correlate with virulence. We have also recently generated complete genome sequences for 6 strains of O. tsutsugamushi, bringing the total number of completely sequenced strains up to 8. These strains will all be used in this study. Our preliminary work and previous reports led us to the hypothesis that strain specific differences in the virulence of O. tsutsugamushi are driven by differences in expression of bacterial virulence-related genes, resulting in either pathogen clearance or inflammatory induced pathogenesis. This hypothesis will be addressed in the following aims. In Aim 1, we will increase the power of our preliminary correlative analysis by quantifying the virulence and gene expression profiles of a total of 8 strains of O. tsutsugamushi, and will use this information to identify bacterial and host factors that robustly associate with virulence. In Aim 2, we will use an in vitro immune reporter assay to probe the role of specific bacterial genes in virulence, focusing on the factors identified in our preliminary studies and in Aim 1. Results of this study will guide future work on developing novel diagnostic tools and therapies against this pathogen.